Compositions and methods for the production of alpha-herpesviruses

ABSTRACT

The present invention relates to virus growth media that improve the yield of alpha-herpesviruses (e.g., HSV-2) grown in cell cultures. The growth media of the invention include two additives, a disaccharide and a lipid mixture, that can be added to serum-free or serum-enriched growth media to improve the efficiency of virus production. The invention further provides methods of producing alpha-herpesviruses (e.g., HSV-2) in such growth media.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a reissue application of U.S. Pat. No. 8,877,492,filed as U.S. application Ser. No. 13/057,788 and issued on Nov. 4,2014, which is the U.S. national stage filing under 35 U.S.C. §371 ofinternational application PCT/US2009/053407, filed Aug. 11, 2009, whichclaims benefit of U.S. Provisional Application No. 61/188,552, filedAug. 11, 2008.

FIELD OF THE INVENTION

The invention provides compositions including growth media that improvethe yield of alpha-herpesviruses (e.g., HSV-2) grown in cell cultures.The growth media of the invention include at least two additives, adisaccharide and a lipid mixture, which can be added to serum-free orserum-enriched growth media to improve the efficiency of virusproduction. The invention further provides methods of producingalpha-herpesviruses (e.g., HSV-2) in such growth media.

BACKGROUND OF THE INVENTION

The alpha-herpesvirus herpes simplex virus type 2 (HSV-2) is the causeof genital herpes, which can be treated symptomatically but not cured.Hallmarks of herpes virus infection include the establishment oflifelong, latent infections that can reactivate to cause one or morerounds of disease, as well as transmission in the absence of symptoms.Transmission of HSV-2 to newborns at the time of delivery may lead todevastating systemic infection with encephalitis. Further,immunocompromised individuals are at increased risk of seriousdisseminated infection. Also, HSV-2 has been shown to increasesignificantly the risk of becoming infected with HIV-1, the virus thatcauses AIDS. The development of vaccines to prevent or treat HSV-2infection is therefore of considerable importance.

The manufacture of virus-based vaccines (e.g., attenuated or inactivatedvirus particles) generally involves the infection of mammalian celllines in vitro with a small quantity of a virus, followed later byharvest of large quantities of progeny virus. The mammalian cells arepropagated in growth media, which are complex solutions containing alarge number of different salts, small organic molecules such ascarbohydrates and lipids, and macromolecules such as peptides andproteins. Optimization of virus production methods is important to thedevelopment of efficiently produced, cost-effective herpes virusvaccines.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides compositions useful forgrowing cells in culture (e.g., monolayer culture). The compositions ofthe invention include growth media including between 5 mM and 500 mM(e.g., about 50 mM and about 80 mM, or between about 50 mM and about 80mM) of a disaccharide (e.g., sucrose), and a lipid mixture (e.g., alipid mixture derived from a plant or that is synthetically produced,and/or including cholesterol or stigmastanol). The cells grown in thecompositions of the invention can be, for example, Vero, MRC-5, BHK,CEM, and LL-1 cells. The disaccharide can be, for example, sucrose,lactose, maltose, trehalose, or cellobiose, or a mixture thereof. In anexample, the compositions also contain methyl-β-cyclodextrin or serum,such as fetal bovine serum (FBS). The compositions of the invention can,for example, increase production of a virus in cells by at least 10%,relative to cells grown in compositions lacking the disaccharide andlipid mixture. The virus grown in the media can be an alpha-herpesvirus,such as herpes simplex virus 1 (HSV-1) or herpes simplex virus 2(HSV-2). In one example, the HSV-2 has one or more inactivatingmutations (e.g., deletions) in U_(L)5 and U_(L)29 (e.g., virus dl5-29),optionally in combination with one or more inactivating mutations (e.g.,deletions) in U_(L)41 (e.g., virus dl5-29-41). Further, the compositionsof the invention can include, in addition to media components, asdescribed herein, cells and/or viruses, as described herein.

In a second aspect, the invention provides methods of increasing virusproduction in cells, involving culturing the cells in a serum-freemedium containing between 5 mM and 500 mM (e.g., about 50 mM or about 80mM, or between about 50 mM and about 80 mM) of a disaccharide (e.g.,sucrose), and a lipid mixture (e.g., a lipid mixture derived from aplant or that is synthetically produced, and/or including cholesterol orstigmastanol), where the medium increases production of a virus in cellsby at least 10% relative to cells grown in a medium lacking thedisaccharide and lipid mixture. In various examples, the cells aremammalian cells, such as Vero, MRC-5, BHK, CEM, or LL-1 cells. Inanother example, the cells (e.g., AV529-19 cells) can support the growthof a replication defective herpesvirus, such as dl5-29, by expression ofherpesvirus polypeptides U_(L)5 and/or U_(L)29. The disaccharide can be,for example, sucrose, lactose, maltose, trehalose, or cellobiose, or amixture thereof. In a further example, the medium containsmethyl-β-cyclodextrin or serum, such as fetal bovine serum (FBS). Themedia used in the methods of the invention can, for example, increaseproduction of a virus in cells by at least 10% relative to cells grownin a medium lacking a disaccharide and lipid mixture. The virus grown inthe medium can be an alpha-herpesvirus, such as herpes simplex virus 1(HSV-1) and herpes simplex virus 2 (HSV-2). In one example, the HSV-2has one or more inactivating mutations (e.g., deletions) in U_(L)5 andU_(L)29 (e.g., virus dl5-29), optionally in combination with one or moreinactivating mutations (e.g., deletions) in U_(L)41 (e.g., virusdl5-29-41).

The invention provides several advantages. In particular, thecompositions of the invention improve the yield of herpes viruses grownin cell cultures, which provides benefits with respect to efficiency andcost of virus production methods.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the average population doubling times of Verocells (strain AV529-19) grown in three different cell culture media(OptiPro SFM, ExCell, and VP SFM AGT), with and without the addition ofsucrose and a lipid mixture.

FIG. 2 is a graph showing the yield of HSV-2 virus dl5-29 (startingMOI=0.1) produced in AV529-19 cells grown in three different cellculture media (OptiPro SFM, ExCell, and VP SFM AGT), with and withoutthe addition of sucrose and a lipid mixture.

FIG. 3 is a graph showing the total yield per flask of HSV-2 virusdl5-29 (starting MOI=0.1) produced in AV529-19 cells grown in threedifferent cell culture media (OptiPro SFM, ExCell, and VP SFM AGT), withand without the addition of sucrose and a lipid mixture.

FIG. 4 is a graph showing the optimization of culture media additives inOptiPro SFM with 1% FBS. The horizontal line at ˜13 pfu/cell indicatesdl5-29 yield in OptiPro SFM (no FBS) with 1× each of sucrose and a lipidmixture. The horizontal line at ˜8.5 pfu/cell indicates OptiPro SFM (noFBS) with no sucrose/lipid mixture additives.

FIG. 5A is a graph showing virus titer from cells grown in threedifferent culture media (OptiPro, DMEM-F12, and Megavir) in the presenceor absence of sucrose and a lipid mixture.

FIG. 5B is a oneway analysis of the data of FIG. 5A focused on the viraltiter in the presence or absence of additives.

FIG. 5C is a oneway analysis of the data of FIG. 5A focused on the viraltiter in cells incubated in the indicated basal media.

FIG. 6 is a graph showing the PFU/cell production of cells incubated inthree different culture media (OptiPro, DMEM-F12, and Megavir) with andwithout sucrose and a lipid mixture.

FIG. 7A is a graph showing viral titer resulting from incubation withthe indicated amounts of sucrose and a lipid mixture in DMEM-F12.

FIG. 7B is a graph showing the interaction profiles of optimum amountsof lipid concentrate and sucrose. The upper right quadrant shows titeras a function of sucrose concentration in the presence and absence of 1×lipid. The lower left quadrant shows titer as a function of lipidconcentration in the presence and absence of 100 mM sucrose.

FIG. 8 is a graph showing PFU/cell production of cells incubated withthe indicated amounts of sucrose and a lipid mixture in OptiPro.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for use in virusproduction. The compositions of the invention include a cell culturemedium supplemented with disaccharide and lipid mixture additives. Thecompositions can be used to culture cells infected withalpha-herpesviruses and, advantageously, result in high viral yields ofsuch cultures. As discussed further below, in one example, thecompositions and methods of the invention are used in the production ofa herpes simplex virus, which can be used, e.g., as a vaccine or as adelivery vector. The compositions and methods of the invention aredescribed in further detail as follows.

The present invention is based on the discovery that the addition of adisaccharide, sucrose, and a lipid mixture to a base cell culture mediumincreases the yield of an alpha-herpesvirus (i.e., HSV-2 strain dl5-29;see below) by over 100% in static cultures. The viral growth media canbe formulated with or without fetal bovine serum (FBS). This result isapplicable to other growth media, including VP-SFM-AGT(Invitrogen/GIBCO) and ExCell (SAFC)(also see below). The effect of thetwo additives is due to increased productivity of virus on a per-cellbasis, and not due to increased cell density per growth vessel. A matrixanalysis of several additive concentrations was performed to ensure thatoptimal concentrations of both additives were identified. The inventionwas first demonstrated in small culture flasks and later extended tolarge 10-layer Nunc cell factories (NCFs). The compositions and methodsof the invention can also be used in bioreactors for large-scale virusproduction (e.g., for commercial viral vaccine production).

As discussed further below, the compositions and methods of theinvention can be used to increase the production efficiency ofalpha-herpesviruses (e.g., HSV-2), such as replication-defective strainsthat can be used in therapeutic or prophylactic vaccines or as deliveryvectors.

Viral Growth Media

The invention provides growth media that contain both a disaccharide(e.g., sucrose) and a lipid mixture that, when used to produce analpha-herpesvirus (e.g., HSV-2), is capable of increasing the viralyield. The disaccharide and lipid mixture can be added to a base cellculture medium suitable for the culture of cells supporting virusgrowth, such as Vero, BHK, and MRC-5 cells. Exemplary base culture mediainclude OptiPro SFM (Invitrogen/GIBCO), ExCell (SAFC), VP SFM AGT(Invitrogen/GIBCO) (FIGS. 1-3), MegaVir (HyClone), DMEM-F12 (SigmaAldrich), and any other medium in which the cells and virus can grow, ascan be tested using standard methods (see, e.g., below). A cell culturemedium can be formulated with additional nutrient rich additives (e.g.,with serum, such as fetal bovine serum) or without such additives (e.g.,a “serum-free” medium such as OptiPro SFM). Exemplary formulations ofgrowth media of the invention are described below.

Disaccharides

Disaccharides for use in the compositions and methods of the inventioninclude, for example, sucrose, lactose, maltose, trehalose, andcellobiose. The disaccharides (e.g., sucrose) can be added to a suitablegrowth medium at a concentration of, e.g., 1 mM, 5 mM, 10 mM, 20 mM, 30mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM,400 mM, 500 mM, or more (or at any amount in ranges between any of theamounts listed above; when ranges are provided herein, they include bothends of the ranges, as well as all amounts between the ends, unlessotherwise indicated). Thus, in specific examples, the amount ofdisaccharide (e.g., sucrose) can be 50 mM, about 50 mM, 80 mM, about 50mM, or between 50 mM and 80 mM. An exemplary method of determiningoptimal concentrations of a disaccharide for use with any given virus orcell line combination is described below. Mixtures of disaccharides canalso be used in any of the compositions or methods of the invention.

Lipid Mixtures

Lipid mixtures used in the present invention can contain cholesterol,stigmastanol, other lipids, or combinations of lipids (including, e.g.,cholesterol and/or stigmastanol). The lipid mixtures can be naturally orsynthetically derived. Naturally derived lipids can be isolated from ananimal (e.g., a mammal, such as a cow) or isolated from a plant (e.g.,soybean). Lipid formulations derived from non-animal sources (e.g.,synthetic lipids or lipids derived from plants) may be preferred in someinstances for use in viral growth media for the production of analpha-herpesvirus (e.g., HSV-2) for use in vivo (e.g., in the treatmentof human patients).

A method of determining effective and suitable concentrations of lipidmixtures for increasing the production of an alpha-herpesvirus HSV-2,strain dl5-29 is described herein (see, e.g., FIG. 4), and can beapplied in the context of other viruses, such as those described herein.Prepared lipid mixtures for use in the compositions and methods of theinvention are known in the art (e.g., Invitrogen/GIBCO 250× CholesterolLipid Mixture). Other lipid mixtures are described by Gorfien et al.,Biotechnol. Prog. 16:682-687, 2000. Other examples of such mixtures thatcan be used in the invention include adult bovine serum (Sigma ChemicalCo., e.g., Catalog No. L-4646), Ex-Cyte lipid I or Very Low Endotoxin(VLE) lipid (Miles Inc., Catalog No. 82-004-7, 82-004-8, and 82-019-1),and soybean lipid extract (Boehringer Mannheim Biochemicals; e.g.,Catalog No. 1074-482; also see Iscove et al., J. Expt. Med. 147:923-933,1979).

Alpha-Herpesviruses

Alpha-herpesviruses are a subfamily of the linear, double-strandedfamily of DNA viruses designated Herpesviridae. Alpha-herpesvirusesinclude the genera Simplexvirus (e.g., ateline herpesvirus 1, bovineherpesvirus 2, cercopithecine herpesvirus 2, human herpesvirus 1 (herpessimplex virus-1; HSV-1), human herpesvirus 2 (herpes simplex virus-2;HSV-2), macacine herpesvirus 1 (previously known as cercopithecineherpesvirus 1), macropodid herpesvirus 1 and 2, papiine herpesvirus 2(previously known as cercopithecine herpesvirus 16), and saimiriineherpesvirus 1), Varicellovirus (e.g., bovine herpesvirus 1, bovineherpesvirus 5, bubaline herpesvirus 1, canid herpesvirus 1, caprineherpesvirus 1, cercopithecine herpesvirus 9, cervid herpesvirus 1 and 2,equid herpesvirus 1, 3, 4, 6 (tentative), 8 and 9, felid herpesvirus 1,human herpesvirus 3 (varicella zoster virus, VZV), phocid herpesvirus 1,and suid herpesvirus 1), Mardivirus (e.g., columbid herpesvirus 1,gallid herpesvirus 2, gallid herpesvirus 3, and meleagrid herpesvirus1), and Iltovirus (e.g., gallid herpesvirus 1, psittacid herpesvirus 1,chelonid herpesvirus 5, and chelonid herpesvirus 6).

A virus grown in the growth medium of the invention can be anyalpha-herpesvirus, or any virus derived therefrom, for example, HSV-1 orHSV-2 strains. An alpha-herpesvirus produced according to the methods ofthe invention can have, for example, the sequence of an HSV-1 or HSV-2genome modified by nucleotide mutations, including deletions andinsertions, such as in the viruses described above and elsewhere herein.In particular, derivatives that can be used in the practice of theinvention include viruses that have genetic mutations, particularlymutations that result in viral attenuation. Examples of such virusesinclude viruses having deletions in U_(L)5, U_(L)29, optionally incombination with U_(L)41, such as the HSV-2 strain dl5-29, which isdescribed further below (see also WO 99/06069; U.S. Patent PublicationNo. 20020009462; DaCosta et al., J. Virology 74:7963-7971, 2000; andU.S. Pat. No. 6,841,373), as well as dl5-29-41 (see, e.g., WO2007/016239).

Additional examples of mutant viruses that can be produced according tothe methods of the invention include strain 1716 (MacLean et al., J.Gen. Virol. 72:631-639, 1991), strains R3616 and R4009 (Chou et al.,Proc. Natl. Acad. Sci. USA 89:3266-3270, 1992), and R930 (Chou et al.,J. Virol. 68(12):8304-8311, 1994), all of which have mutations inICP34.5, strain d120, which has a deletion in ICP4 (DeLuca et al., J.Virol. 56(2):558-570, 1985), strain d27-1 (Rice et al., J. Virol.64(4):1704-1715, 1990), which has a deletion in ICP27), and strain d92,which has deletions in both ICP27 and ICP4 (Samaniego et al., J. Virol.69(9):5705-5715, 1995). Replication competent viruses, such as thosethat contain one or more mutations in ICP10, and replication incompetentviruses, such as those that contain one or more mutations in the ICP8gene, can also be produced according to the methods of the presentinvention.

In addition, viruses including nucleotide substitutions can also beused, for example, viruses containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25,50, 100, 200, 350, 500, or more nucleotide substitutions (as well asranges including or between any of these values). The HSV-1 or HSV-2genome can alternatively or additionally be modified by one or moreinsertions or by an extension at either or both ends. Viral derivativesthat can be produced according to the methods of the invention alsoinclude intertypic recombinants containing DNA from, e.g., HSV-1 andHSV-2 strains. Derivatives typically have at least 70% sequence identityto a parent virus from which it is derived (e.g., HSV-1 or HSV-2), suchas, for example, at least 80%, at least 90%, or at least 95% identity.

In addition to viruses used as vaccines against alpha-herpes virusinfection, the methods of the invention can also be used to produceviruses that can be used in the delivery of heterologous, non-herpesvirus sequences (see, e.g., WO 01/53507 and U.S. Pat. No. 7,118,755).

Herpes Simplex Virus 2 Strain dl5-29

Virus dl5-29 is a replication-defective HSV-2 virus, which containsdeletions of the open reading frames for UL5, a component of thehelicase-primase complex and an essential viral protein for viral DNAsynthesis, and UL29, encoding the ICP8 DNA-binding protein, which isalso an essential protein for viral DNA synthesis (see, e.g., WO99/06069). Virus dl5-29 replicates only in cells engineered tocomplement the two genes, U_(L)5 and U_(L)29, which were deleted fromits genome. The Vero cell line, a transformed cell line derived fromAfrican green monkey kidney cells, is often used as a cell substrate forthe production of various vaccines. After genetic manipulation allowsfor expression of herpes viral genes U_(L)5 and U_(L)29, engineered Verocell strain AV529-19 can be used as a substrate for the production ofvirus dl5-29. Use of the methods of the invention in the production ofdl5-29 is described further below. Further, in addition to dl5-29, asdescribed, for example, in WO 99/06069, the methods of the invention canbe used in connection with other herpes viruses (e.g., HSV-2) havingdeletions in U_(L)5 and U_(L)29 open reading frames, or portionsthereof, which render the viruses dependent upon complementation ofthese proteins for replication. In addition to dl5-29, as describedabove, the methods of the invention can be used in the production ofviruses (e.g., dl5-29-41), which, in addition to having deletions inU_(L)5 and U_(L)29 sequences, also have an alteration (such as one ormore deletions) of U_(L)41 nucleic acid sequences, by which thealteration increases the immunogenicity of the virus. A specific exampleof a virus having deletions in U_(L)5, U_(L)29, and U_(L)41 sequences(dl5-29-41), as well as general teachings of such viruses, are providedin WO 2007/016239.

Cell Lines

Cell lines used in the invention include cell lines that supportalpha-herpesvirus growth, such as Vero (e.g., strain AV529-19), MRC-5,BHK, CEM, and LL-1 cells. A suitable cell line is one that hostsalpha-herpesviruses. Typically, the cell line is a mammalian cell line,such as a rodent, non-human primate (e.g., monkey), or human cell line.In order to allow growth of viruses lacking a gene encoding a proteinessential for viral growth, the host cell line must include a nucleicacid sequence encoding the polypeptide missing from the virus (e.g., adeleted or mutated polypeptide). The host cell line can provide morethan one polypeptide, in trans, to support viral growth (e.g., U_(L)5and U_(L)29 are provided by AV529-19). Such polypeptides includestructural and non-structural (e.g., functional) alpha-herpesviruspolypeptides, which can complement the growth of another virus in whichthe gene for the homologous polypeptide is deleted or otherwise mutated.The use of a Vero cell line to grow replication-incompetentherpesviruses is described in, e.g., U.S. Pat. No. 6,841,373, which isincorporated herein by reference.

Cell lines expressing functional herpes virus structural polypeptidescan be produced by standard methods, such as by co-transfectingmammalian cells, for example, Vero, MRC-5, BHK, CEM, and LL-1 cells,with one or more vectors, such as a plasmid vector, including a nucleicacid molecule encoding the polypeptide(s), and a vector, such as aplasmid vector, encoding a selectable marker (e.g., the neo gene forneomycin/G418 antibiotic resistance). Clones possessing the selectablemarker are then screened further to determine which clones also expressfunctional polypeptide(s) using methods known to those skilled in theart (see, e.g., Rice et al., J. Virol. 64(4):1704-1715, 1990).

Compositions Including Media, Cells, and, Optionally, Viruses

The invention also includes compositions that include a medium of theinvention, in combination with cells for use in virus production,according to the invention. These compositions can also, optionally,include viruses produced in the cells of the invention. Thus,compositions of the invention can contain media that includes adisaccharide (e.g., sucrose, lactose, maltose, trehalose, andcellobiose) at, for example, any of the concentrations noted above, aswell as a lipid mixture (containing, for example, cholesterol and/orstigmastanol; and being synthetic or derived from animal or plant (e.g.,soybean) sources). Specific examples of lipid mixtures and their amountsthat can be included in such compositions are provided above. The mediaof the invention can include, for example, the base culture mediasdescribed above. Further, the media in the compositions of the inventioncan also optionally include components such as serum and/or amino acids,as described above.

Cells included in the compositions of the invention include thosedescribed above, e.g., Vero (e.g., AV529-19), MRC-5, BHK, CEM, and LL-1cells, which may include transgenes encoding polypeptides that may bemutated or otherwise deficient in viruses produced in the cells. Thecells can be plated, in suspension, intact, or in disrupted form (e.g.,after sonication). Viruses included in the compositions of the inventioninclude alpha-herpesviruses, including HSV-2 viruses as well as theother alpha-herpes viruses listed above. The viruses can includeattenuating and/or other beneficial mutations as described herein, suchas are present in viruses dl5-29 and dl5-29-41. The compositions of theinvention can be used in methods for producing viruses as describedherein.

Production of HSV-2 Strain dl5-29 Using Media Additives

The production of an HSV vaccine virus including deletions in U_(L)5 andU_(L)29 (e.g., dl5-29) can be performed using a complementary Vero cellline such as AV529-19, which is genetically engineered two express twogenes, U_(L)5 and U_(L)29, that are necessary to support virus growth.Typically, viral yields have been below 10 pfu per cell, and methodswere sought to attempt to boost the yield. The addition of sucrose and alipid mixture to the growth and infection media improved yields toapproximately 15 pfu per cell in preliminary studies using T-flasks anda limited number of NUNC Cell Factories (NCFs). Based upon thesestudies, optimized concentrations of sucrose and the lipid mixture wereadded to current media formulations on a larger scale and a productionrun utilizing four NCFs was performed to mimic a significant portion ofa production run designed to produce viral seed or vaccine product forpreclinical and clinical studies. The cells were seeded onto NUNC CellFactories in OptiPro SFM media containing 10% Fetal Bovine Serum, 4 mML-Glutamine, G418, and supplemented with 50 mM sucrose and 0.5× of a250× concentrated lipid mixture (Invitrogen/GIBCO 250× cholesterol lipidconcentrate) and grown for approximately 96 hours at 37° C. and 5% CO₂.Infection media consisted of the same OptiPro media, only with 1% FBSand without G418. Infections were at an estimated MOI of 0.01, andfollowing approximately 72 hours incubation at 34° C. and 5% CO₂, NCFswere harvested. Aliquots were drawn from each unit for sonication andviral titer by standard plaque assay, and bulk harvests were centrifugedand pelleted in stabilization buffer for downstream processing. Plaqueassay results indicated total viral yield for each NCF of about 2×10¹⁰pfu, with an average of 19 pfu/cell. This is more than a two-foldincrease in viral yield compared to previous runs without the sucroseand cholesterol lipid concentrate additives. Viral yields between NCFswere consistent, ranging from 18 to 20 pfu/cell.

Identification of Optimal Media Additive Concentrations

Preliminary studies were performed in T-25 flasks to determine optimalconcentrations of sucrose and a lipid mixture in growth and infectionmedia during production of dl5-29 virus. This experiment to optimizeadditive concentrations consisted of a matrix of samples inconcentrations of 0.2×, 0.5×, 1.0×, 2.0×, and 5.0× for each additivewith 1× equivalent to 50 mM sucrose or 1× of a 250× cholesterol lipidconcentrate. Raw data plaque counts were compiled onto an EXCELspreadsheet, in which raw titers were calculated by multiplying theaverage plaque count by its dilution factor and dividing that quantityby 0.2. The resulting number was then multiplied by 5 to reflect theoriginal 1/5 dilution of the sonicated sample to determine the pfu/mL.Standard deviations and % CV were also calculated to assist indetermining the validity of the data. Data collected from this study areshown in FIG. 4, plotting calculated pfu/cell versus additiveconcentrations. All data shown are from growth and infection mediacontaining serum, except the two horizontal lines at ˜8.5 pfu/cell and13 pfu/cell that indicate serum-free media without and with additives,respectively. The data indicate a substantial drop in titer withcholesterol concentration above 1×. The highest titers were achievedwith 1× sucrose, 0.5× cholesterol and 2× sucrose, 1× cholesterol. Basedupon these data, and for ease of formulation, the optimum concentrationsused to go forward were sucrose at 1× (50 mM) and 0.5× of a 250×cholesterol lipid concentrate.

Confirmation of Additive Effect at Larger Scale

Based upon optimization study results, media for growth and infectionwere prepared for use in NCFs to compare the effects of these additiveson a larger scale format. Two NCFs were prepared, one using media withadditives (50 mM sucrose, 1× cholesterol lipid concentrate), the otherwithout as a control. Results of this study are shown in Table 1.

TABLE 1 Comparison of dl5-29 yields of NCF treated or not treated withadditives. Avg. Titer Total pfu/ Passage (pfu/mL) pfu/NCF pfu/cm² celldl5-29 NCF with 29 1.26E+07 1.26E+10 2.00E+06 12.63 additives dl5-29 NCFwithout 29 6.08E+06 6.08E+09 9.63E+05 6.08 additives

Further analysis was performed to calculate the viral yield per cm², perNCF, and per cell. These calculations were based upon the total cm² ofthe NCF as 6320, and an estimation of a confluent NCF as having 1.0×10⁹total cells. In addition, passage numbers for the AV529-19 cells areshown at the time of infection. Results indicate a benefit from thesucrose and cholesterol additives. Pfu/cell data were consistent with,or slightly lower than previous studies, however, the passage number ofthe cells used was at the higher limit of optimum use.

Results from a pilot study utilizing 4 NCFs are shown on Table 2. Theresults show nearly 2.0×10¹⁰ total pfu/NCF, with yield efficiencies of18-19 pfu/cell. Yields were consistent throughout all four NCFsevaluated in this pilot study.

TABLE 2 Calculated average viral titers (pfu/mL), total pfu/NCF,pfu/cm², and pfu/cell of NCF viral harvests. Avg. Titer Total pfu/Passage (pfu/mL) pfu/NCF pfu/cm² cell dl5-29 NCF 1 19 1.93E+07 1.93E+103.05E+06 19.29 dl5-29 NCF 2 19 1.82E+07 1.82E+10 2.88E+06 18.21 dl5-29NCF 3 19 1.97E+07 1.97E+10 3.12E+06 19.71 dl5-29 NCF 4 19 1.94E+071.94E+10 3.07E+06 19.38

Based on the results provided above, the best upstream yields obtainedso far are about 1.9×10¹⁰ pfu/NCF, corresponding to an average of 19pfu/cell. This is more than a two-fold increase in viral yield ascompared to previous runs without sucrose and cholesterol lipidconcentrate. In the four-NCF pilot study, viral yields between NCFs werevery consistent, ranging from 18 to 20 pfu/cell. The optimal conditionsdescribed here yield useful amounts of virus.

Additional studies were carried out using OptiPro, DMEM-F12, and Megavirmedia (see FIGS. 5A-8). Oneway analysis of the titer production by basalmedia type shows that Megavir and DMEM-F12 titers have a largerimprovement on virus production with sucrose and cholesterol thanOptiPro. This analysis also suggests that the titer improvement inOptiPro, under certain conditions, may not be statistically significant(FIGS. 5A-5C and FIG. 6).

Sucrose, as a single factor, has an impact on virus production, butcholesterol by itself does not improve the virus production (FIGS. 7Aand 7B). The simultaneous presence of sucrose and cholesterol has asignificant impact on virus production. To maximize the titer, the datasuggest 1× cholesterol and 80 mM sucrose in DMEM-F 12 to be the optimalcondition for viral production. Observed increases in viral productionare partially due to an increase in the specific production rate of thecells, showing an increase in the presence of the sucrose andcholesterol from 57.9 PFU/cell to 85.3 PFU/cell (FIG. 8).

Methods

Design of the 4 NCF Pilot Study (Table 2)

The purpose of this study was to scale-up production of herpes simplexvirus strain dl5-29 from infection of Vero (AV529-19) cells in four Nunccell factories (NCFs; Nalge-Nunc). The cells can be grown in OptiPro SFMmedia (Invitrogen/GIBCO, catalog no. 12309-019) containing 10% fetalbovine serum (FBS; JRH, catalog no. 12106-500M), 4 mM L-glutamine(Invitrogen/GIBCO, catalog no. 25030-081), G418 (Invitrogen/GIBCO,catalog no. 10131-027), and supplemented with 50 mM sucrose (EMD,catalog no. SX1075-1) and 0.5× of a 250× cholesterol lipid concentrate(Invitrogen/GIBCO, catalog no. 12531-018). Infection media consisted ofthe same OptiPro media containing 1% FBS but lacking G418. Cell cultureswere infected with an approximate MOI of 0.01, and allowed to incubateapproximately 72 hours at 34° C. and 5% CO₂. Following incubation, NCFswere harvested. Aliquots were drawn for each unit for sonication andviral titer by standard plaque assay (described below), and bulkharvests were centrifuged and pelleted in stabilization buffer fordownstream processing.

Preparation of 1 M Sucrose Concentrate in Optipro (400 mL)

Mix the following reagents and warm to 37° C.:

-   -   sucrose (136.92 g)    -   OptiPro SFM (200 mL)    -   FBS (10%; 40 mL)    -   L-glutamine (8 mL)    -   G418 (4 mL)    -   cholesterol lipid concentrate (0.8 mL)        When dissolved, QS volume to 400 mL total with OptiPro SFM, then        filter sterilize (0.2 μm).        Preparation of Seeding Media for NCFs (Per L)

Mix the following reagents:

-   -   OptiPro SFM (868 mL)    -   FBS (10%; 100 mL)    -   L-glutamine (20 mL)    -   G418 (10 mL)    -   cholesterol lipid concentrate (2 mL)        When mixed well, remove 50 mL of media mixture and replace with        50 mL of 1 M sucrose concentrate in OptiPro SFM. The final        mixture will have a sucrose concentration of 50 mM.        Preparation of 1 M Sucrose Concentrate in OptiPro (200 mL for        Infection Media)

Mix the following reagents and warm to 37° C.:

-   -   sucrose (68.46 g)    -   OptiPro SFM (100 mL)    -   FBS (1%; 2 mL)    -   L-glutamine (4 mL)    -   cholesterol lipid concentrate (0.4 mL)        When dissolved, QS volume to 200 mL total with OptiPro SFM, then        filter sterilize (0.2 μm).        Preparation of Infection Media for NCFs (Per L)

Mix the following reagents:

-   -   OptiPro SFM (968 mL)    -   FBS (1%; 10 mL)    -   L-glutamine (20 mL)    -   cholesterol lipid concentrate (2 mL)        When mixed well, remove 50 mL of media mixture and replace with        50 mL of 1 M sucrose concentrate in OptiPro. The final mixture        will have a sucrose concentration of 50 mM.        Preparation of 10% Sucrose in 1× Stabilization Buffer

Mix the following reagents:

-   -   sucrose (10 g)    -   10× Stabilization Buffer (10 mL)        Add RODI to bring total volume to 100 mL. Mix well and then        place in a 37° C. waterbath for approximately 30 minutes to        fully dissolve. Filter-sterilize using a 0.2 μm sterile filter        unit.        NUNC Cell Factory Seeding Procedure

Harvest 8 triple flasks of Vero cells (AV529-19) grown to approximately90% confluence in OptiPro medium containing 10% FBS with 4 mML-glutamine, and G418. Following harvest, draw off a 1 mL aliquot forcell count using a Vi-Cell instrument. Under aseptic conditions, seedNCFs at 10⁸ cells/NCF using pre-infection media (OptiPro SFM with 10%FBS+4 mM L-glutamine, G418, 50 mM sucrose and 0.5× cholesterol lipidmixture; 2 L). Grow to near confluence (approximately 3-4 days) in anincubator at 37° C. and 5% CO₂.

NUNC Cell Factory Infection Procedure

Pour off old media and rinse NCF with 500 mL DPBS and remove. Infect atfull volume infection media (1 L) with dl5-29 at a MOI of 0.01 asfollows, assuming 10⁹ cells per confluent NCF: NCF #1-4: 10⁷ pfu/NCF ofHSV98 (dl5-29) in infection media (OptiPro SFM with 1% FBS+4 mML-glutamine, 50 mM sucrose and 0.5× cholesterol lipid concentrate).Based on a stock titer of 1.6×10⁸ pfu/mL, add 625 μL of virus stock perNCF. Incubate the NCFs at 34° C. for 72 hours or until approximately100% CPE is evident.

NUNC Cell Factory Harvest Procedure

When 100% CPE (nearly 100% of cells rounded up and easily detached) isevident, tap NCFs several times to dislodge all cells from monolayer.With 100% CPE, cells should already be mostly detached. Aseptically pouroff infection media from NCF into a sterile container. Mix well, thendraw off 1 mL aliquot and add to sterile 15 mL centrifuge tubecontaining 3.75 mL of OptiPro SFM with 10% FBS and 4 mM L-glutamine, and250 μL of HSA. This is used as a 1:5 dilution of the NCF harvest and isto be sonicated and titered to determine viral yield. Divide the NCFharvest equally into 4×250 mL sterile centrifuge tubes. Spin incentrifuge at 1000 RPM for 10 minutes at 4° C. Decant resultingsupernatants and save in 1 L sterile bottle labeled as conditionedmedia. Store bottles at 4° C. Using 20 mL of 10% Sucrose in 1×Stabilization buffer, aseptically resuspend and combine pellets in BSC.Quick-freeze combined pellet and 1 mL centrifuge tube containing titersample in ethanol/dry ice bath until frozen solid. Then place samples in−80° C. Freezer.

Sonication Procedure

Remove 15 mL centrifuge tubes containing titer samples and thaw quicklyin a 37° C. waterbath. Immediately after thawing, place on ice.Aseptically sonicate cell suspension sample using probe sonicator;Outlet level 3, Duty cycle 40% for approximately 15 seconds/mL on ice(total of 75 seconds). Spin down sonicated suspension samples incentrifuge at 5000 g for 10 minutes at 4° C. to remove cell debris.Aseptically pour off supernatant into sterile, labeled 15 mL centrifugetubes. Pellets are discarded and proceed directly to viral titerprocedure.

Virus Infectivity Assay (Plaque A, Assay)

Test samples were tested in 12-well plates that were seeded with Verocells (4×10⁵ cells/well) for virus infectivity and quantification. Theywere serially diluted 10-fold in duplicate in minimum essential medium(MEM) containing 1% FBS supplemented with 100 units/mL penicillin and100 μg/mL streptomycin, and L-glutamine (completed cell culture medium).Dilutions were then plated onto the cells at 200 μL/well in triplicatewells. A positive control is also serially diluted and plated. Theinoculated plates were incubated at 37° C. with 5% CO₂ for 60 minuteswith rocking of the plates every 15 minutes to allow virus adsorption.One mL of completed medium with 0.8% methyl cellulose was added to eachwell and the plates were returned to incubation at 37° C. with 5% CO₂for 2 days. Medium was then removed from all wells and approximately 1mL of 1% crystal violet in 70% methanol was added to each well to fixand stain plates. Plates were left at room temperature for more than 60minutes to stain cells, then the stain was removed, wells were washedwith tap water, and the plates were air-dried. Viral plaques werecounted using a stereomicroscope with plates held over a light box.Virus concentrations are expressed as pfu/mL. Further calculations arethen made to determine the total viral yield per NCF, viral yield/cm²,and viral yield/cell.

Other Embodiments

All references, publications, patent applications, and patents cited inthis specification are herein incorporated by reference as if eachindividual publication or patent were specifically and individuallyindicated to be incorporated by reference. Use of singular forms herein,such as “a” and “the,” does not exclude indication of the correspondingplural form, unless the context indicates to the contrary. Although theinvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof the invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

Other embodiments are within the following claims.

What is claimed is:
 1. A composition comprising (i) a growth mediumsupplemented with between 5 mM and 500 mM of a disaccharide and a lipidmixture, (ii) Vero cells that express herpes simplex virus 2 (HSV-2)polypeptides U_(L)5 and U_(L)29, and (iii) an HSV-2 having inactivatingmutations in U_(L)5 and U_(L)29.
 2. The composition of claim 1, whereinsaid disaccharide is sucrose, lactose, maltose, trehalose, orcellobiose, or a mixture thereof.
 3. The composition of claim 1, whereinsaid disaccharide is present in said medium at 50 mM or 80 mM, orbetween 50 mM and 80 mM.
 4. The composition of claim 1, wherein saidmedium further comprises methyl-β-cyclodextrin and/or serum.
 5. Thecomposition of claim 4, wherein said serum is fetal bovine serum (FBS).6. The composition of claim 1, wherein said lipid mixture is from aplant, is synthetic, comprises cholesterol, and/or comprisesstigmastanol.
 7. The composition of claim 1, wherein said mediumincreases production of said HSV-2 in the cells by at least 10% relativeto HSV-2 production in cells grown in a medium lacking said disaccharideand said lipid mixture.
 8. A method of increasing herpesvirus productionin cells, said method comprising culturing the composition of claim 1,wherein said composition increases production of the herpesvirus by atleast 10% relative to a herpesvirus cultured in cells grown in a mediumlacking said disaccharide or said lipid mixture, wherein saidherpesvirus is a replication-defective herpesvirus having inactivatingmutations in U_(L)5 and U_(L)29, optionally in combination with aninactivating mutation in U_(L)41.
 9. The method of claim 8, wherein saidcells are AV529-19 cells.
 10. The method of claim 8, wherein said cellsexpress the herpesvirus polypeptides U_(L)5 and/or U_(L)29.
 11. Themethod of claim 8, wherein said disaccharide is sucrose, lactose,maltose, trehalose, or cellobiose, or a mixture thereof.
 12. The methodof claim 8, wherein said disaccharide is present at 50 mM or 80 mM, orbetween 50 mM and 80 mM.
 13. The method of claim 8, wherein said mediumfurther comprises methyl-β-cyclodextrin and/or serum.
 14. The method ofclaim 13, wherein said serum is fetal bovine serum (FBS).
 15. The methodof claim 8, wherein said lipid mixture is from a plant, is synthetic,comprises cholesterol, and/or comprises stigmastanol.
 16. The method ofclaim 8, wherein said method increases production of a herpesvirus inthe cells by at least 10% relative to herpesvirus production in cellsgrown in a composition lacking said disaccharide and said lipid mixture.17. The composition of claim 1, wherein said HSV-2 has an inactivatingmutation in U_(L)41.
 18. The composition of claim 1, wherein said HSV-2is strain dl5-29.
 19. The composition of claim 18, wherein said Verocells are AV529-19 cells.
 20. A composition comprising (i) a growthmedium supplemented with between 5 mM and 500 mM of a disaccharide and alipid mixture, (ii) Vero cells engineered to express herpes simplexvirus polypeptides U_(L)5 and U_(L)29, and (iii) a herpes simplex virus2 (HSV-2) having inactivating mutations in U_(L)5 and U_(L)29.
 21. Thecomposition of claim 20, wherein said disaccharide is sucrose, lactose,maltose, trehalose, or cellobiose, or a mixture thereof.
 22. Thecomposition of claim 20, wherein said disaccharide is present in saidmedium at 50 mM or 80 mM, or between 50 mM and 80 mM.
 23. Thecomposition of claim 20, wherein said medium further comprisesmethyl-β-cyclodextrin and/or serum.
 24. The composition of claim 20,wherein said lipid mixture is from a plant, is synthetic, comprisescholesterol, and/or comprises stigmastanol.
 25. The composition of claim20, wherein said medium increases production of said HSV-2 in the cellsby at least 10% relative to HSV-2 production in cells grown in a mediumlacking said disaccharide and said lipid mixture.
 26. The composition ofclaim 20, wherein said HSV-2 is strain dl5-29.
 27. The composition ofclaim 20, wherein said Vero cells are AV529-19 cells.